Abstract The overall goal of this project is to understand the process of ribosome assembly in bacterial cells. The ribosome is the molecular machine that is responsible for protein synthesis in all cells, and biosynthesis of ribosomes is a fundamentally important aspect of cell physiology. We are taking a broadly based biophysical approach to probe the mechanism of ribosome assembly both in cells, and in vitro. Key questions to be addressed include understanding the RNA folding events that underlie the assembly process, understanding the role of assembly cofactors, and understanding the role of co-transcriptional assembly. Approaches to be applied to these questions include quantitative mass spectrometry to analyze the composition of assembly intermediates, cryo-electron microscopy to understand the structures of the assembly intermediates, and single molecule fluorescence methods to understand the dynamics of assembly intermediates. Data from these diverse approaches will be synthesized into a dynamic model for the process of how a ribosome is assembled in cells.